Administration of spironolactone (SL), an antihypertensive and diuretic drug, causes a reduction in the formation of testosterone in the testes of various animal species. The reduction in androgen formation results from a concomitant loss of testicular cytochrome P-450 (P-450) and of testicular 17 alpha-hydroxylase activity. A similar loss of P-450 occurs in the adrenals of those animals which produce cortisol rather than corticosterone. The mechanism by which SL decreases P-450 in the testis or adrenal is obscure. A plausible explanation for the loss of P-450 is one in which SL causes a destruction of the heme of P-450 associated with steroid 17 alpha-hydroxylation. In this mechanism SL or a metabolite of SL would be catalyzed by P-450 to an active metabolite that would, in turn, be responsible for the destruction of the heme of P-450. Such a mechanism would explain the loss of heme that accompanies the loss of testicular or adrenal P-450. This grant proposal is aimed, therefore, at answering the following questions: (1) What is the mechanism by which SL causes the loss of P-450 in adrenal or testicular tissues? (2) Can the administration of SL alter the plasma concentration of other steroids besides testosterone or estradiol? and (3) Can the administration of SL alter the hydroxylating activity of other P-450 dependent enzymes in the adrenal or testis, or is the loss of P-450 by SL specific for the P-450 associated with steroid 17 alpha- hydroxylation? High Pressure liquid chromatography (HPLC) and mass spectrometry will be used for the isolation and identification of microsomal metabolites either from the breakdown of radiolabeled heme or from the metabolism of radiolabeled SL. The plasma concentration of steroids will be determined by radioimmunoassay. The specific activity of testicular or adrenal hydroxylating enzymes will be determined from their mitochondrial or microsomal rate of conversion of radiolabeled substrates to radiolabeled metabolites. HPLC or TLC methods will be employed for the isolation of the steroid metabolites. Cytochrome P-450 will be measured spectrophotometrically.